The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress. Mutations within the p53 gene have been well documented in more than half of all human tumors. Accumulating evidence further indicates that, in the cells that retain wild-type p53, other defects in the p53 pathway also play an important role in tumorigenesis. Tight regulation of p53 is essential for its effect on tumorigenesis as well as maintaining normal cell growth. The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Although the mammalian homolog of Sir2 has also been shown to contain a histone deacetylase activity, its in vivo targets as well as biological function remain unclear. Recently, we have shown that mammalian Sir2a physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2a, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2a represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2a point-mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a novel p53 regulatory pathway mediated by mammalian Sir2a. The overall objective of this project is to demonstrate the precise role of Sir2a in apoptosis and cell senescence, and elucidate this novel p53 regulatory pathway in the stress response. The first specific aim is to elucidate the role for Sir2a in tile regulation of cell senescence and apoptosis. We will examine the role for Sir2a in both oxidative stress- and oncogene ras-induced cell senescence, and also identify key gene targets for Sir2a in the regulation of cell senescence vs. apoptosis based on gene expression profiling. The second aim is to identify novel regulatory factors for mammalian Sir2a. Biochemical purification and identification of human Sir2a-associated protein complexes will be performed and the functional consequence of these novel interactions will be tested. The third aim is to define the physiological role of mammalian Sir2a in vivo. We will develop a mouse model for overexpression of Sir2a or the Sir2a dominant negative mutant to assess for tumor and other phenotypes (e.g. ageing). We will also examine the stress response (DNA damage and oxidative stress) in the cells derived from transgenic mice and test the role of Sir2a in replicative senescence of the primary cells derived from the mice.